The overall goal of the proposed research is to develop, validate, and apply electron paramagnetic resonance (EPR) methodology for measuring distances in biological systems. EPR studies can be performed in disordered solids or in solutions and even in whole cells. EPR is particularly important for characterizing membrane-bound proteins that are difficult to crystallize. The sites to be studied by EPR can be naturally-occurring metal ions or radicals, or spin labels or metals attached at selected locations introduced by site-directed mutagenesis. Even a few measurements of longer distances can provide key information to define the three-dimensional structure of a large protein or assembly of proteins. The larger magnetic moment of an unpaired electron than of a nuclear spin permits measurement of longer distances by EPR than by nuclear magnetic resonance (NMR). The emphasis of the proposed research is on pulsed EPR methods to determine distances longer than about 20 Angstroms, which is approximately the upper limit for EPR methods based on continuous wave lineshapes. We propose to use the distance-dependent changes in electron spin relaxation rates to determine the distance between a rapidly relaxing electron spin such as iron(III) and a nitroxyl spin label. These methods will be calibrated with spin-labeled variants of metmyoglobin of known structure. The X-ray crystal structure of the iron transport protein, iron protein A, did not define the binding site of the iron siderophore, iron enterobactin. We propose to use our pulse techniques to determine the location of the iron binding site. Our model predicts that experiments at lower microwave frequency will permit measurements of longer distances than are accessible at 9.2 GHz and we propose to test that prediction by performing measurements at 2.5 GHz. Current pulsed EPR measurements of distances are performed at cryogenic temperatures. We will test the feasibility of measurements in fluid solution. For doubly spin-labeled samples of human carbonic anhydrase II, T4 lysozyme, and iron protein A we propose to develop and test pulsed methods to determine spin-spin distances greater than about 20 Angstroms.